Linear sucrose gradients

If the expression of the mRNA results in a clear signal, a linear sucrose gradient (6-20% sucrose, 5 mM EDTA, 0.25% (w/v) sarcosyl, 15 mM PIPES-NaOH, pH 6.4) is prepared in SW27 tubes or equivalent and the mRNA is carefully loaded on top. After centrifugation (19 h, 80,000 xg), about 33 fractions of 1 ml each are collected, and the mRNA is precipitated using sodium acetate and ethanol. Prior to injection, the RNA of each fraction is pelleted, washed and dissolved in RNAse-free water as described above. Upon injection of 50 ng fractionated mRNA, a two- to fivefold increase in transport activity is expected for the positive mRNA-fraction compared with unfractionated mRNA. All other fractions should not show any specific functional activity. In cases where two neighbouring fractions induce transport activity, pooling of these should be considered. 

Figure 5. Size analysis of Inhibitors I and 11 specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. Poly(A ) RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf polyfA)" RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly...

Figure 6. Partial purification of Inhibitors I and II mRNA. Fractions containing Inhibitors I and II mRNA determined by in vitro translation analyses were recovered from an initial 15-30% linear sucrose gradient, precipitated by cold ethanol, and applied to a 10-25% linear sucrose gradient. The sample was centrifuged for 36 h at 25,000 rpm. Fractions of the gradient were collected and subjected to in vitro translation analyses. The upper graph represents total methionine incorporation assayed with 1 jiL of the translation mixture as described (ll). The bottom figure quantitates the radiolabel incorporated specifically into Inhibitor I (solid bars) and Inhibitor II (open bars).

Figure 9-28. Frequency distribution of equilibrium densities of mitochondria (cytochrome oxidase), peroxisomes (urate oxidase), and lysosomes (acid phosphatase). Mitochondrial fractions from rat liver equilibrated in a gradient of glycogen (initially linear from 0 to 30.6 g/lOO ml) in 0.5M sucrose and in a linear sucrose gradient (59.7-117.0 g/100 ml). [From H. Beaufay et al., Biochem. /., 92 184 (1964).]...

For isolelectric focusing the sample is mixed into a linear sucrose gradient from 35 to 0% (w/v), containing 5% (by volume) of carrier ampholyte solution, pH 4—7, (equivalent to an ampholyte concentration of 2%, w/v) in a 110-ml electrofocusing column (LKB, Sweden). The cathode space is filled with 1.5% (w/v) of ethylene diamine in a 50% (w/v) sucrose solution the sample space is topped off with 0.1% aqueous H2SO4 as anode solution. Focusing is performed for 72 h at 500 V then the contents are collected in 3-ml fractions. 

Etioplast and thylakoid membranes were isolated in 50 mM Tricine (pH 7.6) containing 17% sucrose, 2 mM MgC12 and 1 mM EDTA. The membrane pellets were applied to the linear sucrose gradient (20-60 % w/v). The plastid band was removed and osmotically shocked with isolation buffer minus sucrose. 

Fig. 6. Separation of glyoxysomes (marker enzyme, isocitrate lyase) and mitochondria (marker enzyme, fumarase) on a linear sucrose gradient. For this preparation the total homogenate from endosperm tissue (4-day-old seedlings) was applied to the sucrose gradient. The very low enzyme activities in the upper parts of the gradient show that essentially all the mitochondria and glyoxysomes have been recovered intact (data of Kagawa el al., 1973).

Supernatant fractions from the basic subcellular fractionation were further separated on a linear sucrose gradient. A 14 ml 10-40% (w/w) sucrose gradient in 50 mM HEPES pH 7.2, 90 mM KCL was prepared with a 65 % (w/v) sucrose cushion. Supernatants (1 ml) were loaded on to... 

The particle size limit is estimated by equation (4) however, this equation does not give the particle size distribution within the floating chylomicron fraction. Pinter and Zilversmit (1962) and Zilversmit (1963) have described an important and readily applied method for estimating the particle size distribution. They employed ultracentrifugal-flotation in concentrated sucrose gradients. Linear sucrose gradients were prepared so that density and viscosity at a point within the gradient were described by the equations ... 

A new system (seven-day-old etiolated leek seedlings) has been employed to study the Intermembrane transfer of lipids and fatty acids in vivo. By vivo pulse-chase experiments, followed by membrane fractionation on linear sucrose gradients, intermembrane transfer events of VLCFA between the light fraction and the heavier ones was demonstrated. 

However, these results did not establish that VLCFA were transferred to the plasmalenuna. The characterization of the membranes obtained after linear sucrose gradients using marker enzymes showed that the heavy membrane fraction contains almost all of the membrane vesicles originating from the plasma membrane, but also that it is relatively heterogeneous. 

The purification of the plasmalemma was undertaken by this method, using either the heavy membrane fraction obtained on linear sucrose gradients, or the microsomal pellet. 

Fig. 16. Effect of ribosome binding on mitochondrial density. Mitochondria, washed with 2 mM EDTA, were incubated for 15 min at 30°C with 0.46, 0.92, 1.84, 3.68, and 5.52 Azeo units of [ H]-labeled cytochrome ribosomes, B through D, respectively. The incubation mixtures were chilled to 0 C and layered on 40-70% linear-sucrose gradients and centrifuged for 1 h at 243,000gmax in a Beckman SW50-1 rotor. The gradients were fractionated and assayed for cytochrome oxidase activity ( — ), radioactivity (A—A), and density (O-—O). Panel A shows the density of mitochondria without added ribosomes.

Fig. 17. Effect of various conditions on mitochondrial density. Mitochondria were sedimented to equilibrium on linear sucrose gradients. After fractionation, the gradients were analyzed for cytochrome oxidase activity and density. (A) Mitochondria from growing cells. (B) Mitochrondria from cells starved for 1 h. (C) Mitochondria from growing cells washed with 2 mM EDTA. (D) Same as (C), except mitochondria were incubated with PH]-labeled ribosomes before centrifugation. (E) Mitochondria from growing cells. Culture was pretreated with 200 Mg/ml cycloheximide prior to harvesting. (F) Mitochondria from growing cells treated with 200 Mg/ml cycloheximide, then starved for 1 h.

Fig. 20. Effect of KCl and puromycin on the release of ribosomes from mitochondria. Mitochondria isolated from growing cells were incubated for 15 min at 30°C with increasing concentrations of KCl, with and without 2.1 mM puromycin as indicated. The incubation mixtures were layered onto 15-30% linear sucrose gradients and were centrifuged for 2 h at 221,800gniax- Under these conditions, the mitochondria were pelleted, and the released ribosomes remained in the gradient and were quantitated by UV flow scanning. (From Kellems et al )...

Fig. 5. Isopycnic banding of membranes. An exponentially growing culture of B42 was filtered, washed, and resuspended in medium with PHJglycerol ( ) or [ CJleucine with no glycerol (O). Membranes were prepared after 1 h of growth. A mixture of the two membrane preparations was layered onto a linear sucrose gradient and centrifuged at 25,000 rpm for 2 h —---, density (g/ml). ...

FIGURE 3. Localization of the envelope ATPase, Inner and outer envelope membranes were resolved on a 0.6 M to 1.2 M linear sucrose gradient as described by Cline et al. (1981). Envelope membranes were prepared as described in Materials and Methods. Gradients were centrifuged in a Beckman SW27 rotor at 24,000 rpm for 14 h. Gradients were fractionated from the bottom and assayed for ATPase,... 

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